Isothermal inactivation of Mycobacterium avium subsp. paratuberculosis in curd simulating the stretching phase in pasta-filata cheese process.

Raw milk and dairy products are usually considered the major sources of Mycobacterium avium subsp. paratuberculosis (MAP) exposure for humans. During the production process of mozzarella cheese, as well as of other pasta-filata cheeses made with pasteurized or raw milk, curd is heated and stretched by addition of hot or boiling water. This step is the critical point for the inactivation of MAP during the production process, but, to our knowledge, no studies have been published about the thermal death time values of MAP in curd. The aim of this study was to determine the inactivation kinetics of MAP in curd used to produce pasta-filata cheese in six independent experiments. The milk was inoculated with a mix of MAP strains (field and registered strains) and, with the aim to simulate the thermal treatment of the curd during the stretching step, samples of 10 g of contaminated curd were vacuum packed and treated separately at six different temperatures from 60°C to 75°C in a water bath. MAP survival was then evaluated by plate count method and inactivation parameters were estimated for determining the thermal resistance of the pathogen directly in the curd. D-values increased from 0.15 min (D75-value) to 4.22 min (D60-value) and the calculated z-value was 10.2°C. These data aid: (i) to design food thermal process treatments defining acceptance limits of critical control points to ensure safety against MAP; (ii) to predict the time/temperature combinations needed to obtain a certain MAP log reduction during the curd stretching step; (iii) to optimize or validate pasta-filata cheese process.

Inactivation of Listeria monocytogenes and Salmonella spp. in Milano-Type Salami Made with Alternative Formulations to the Use of Synthetic Nitrates/Nitrites.

During the manufacture of Italian salami, a traditional meat product, a sequence of hurdles like meat fermentation, air-drying, and long ripening processes are generally sufficient to inhibit the growth of most pathogens. Furthermore, Italian salami are traditionally produced by adding synthetic nitrates/nitrites to raw meat with safety and technological aims, even if controversial opinions about their use still remain, particularly in relation to the consumer demand for natural food products. In this context, the aim of the study was to investigate the inactivation of Listeria monocytogenes and Salmonella spp. during the manufacturing process of Milano-type salami made with different formulations to evaluate the contribution of the hurdles and the vegetable or synthetic additives on the inactivation of pathogens. Thus, a challenge study was performed dividing ca. 400 kg of Milano-type salami batter into three batches: Batch (A) without nitrates/nitrites; Batch (B) with vegetable nitrates, and Batch (C) with synthetic nitrates/nitrites. The batches were separately inoculated with L. monocytogenes and Salmonella spp. and the pathogens' survival was evaluated during the fermentation, draining, and 70-day ripening of the Milano-type salami. The pathogen counts decreased in all tested conditions, even though the highest inactivation of L. monocytogenes and Salmonella spp. (p < 0.05) was observed when nitrates or nitrites were added to the batter. This study shows how the safety of these products cannot exclude the aspect of the hurdle technology during the process, which plays a major role in the reduction of pathogens, but additives like nitrates and nitrites allow for a greater margin of safety. Thus, further studies are needed to validate the use of natural compounds as alternatives to conventional preservatives in meat products. These results may provide new information to support food business operators in producing traditional foods with alternative preservatives and competent authorities in verifying the safety of the products made with natural compounds, and to control the process parameters responsible for the synergistic effect against pathogens such as L. monocytogenes and Salmonella spp.

One-Day Molecular Detection of Salmonella and Campylobacter in Chicken Meat: A Pilot Study.

Salmonella and Campylobacter ssp. are bacterial pathogens responsible for most foodborne infections in EU countries. Poultry serves as a reservoir for these pathogens, and its important role in the meat industry makes it essential to develop a rapid detection assay able to provide results in one day. Indeed, the rapid identification of foodborne pathogens is an important instrument for the monitoring and prevention of epidemic outbreaks. To date, Salmonella and Campylobacter screening is mainly conducted through molecular methods (PCR or real-time PCR) performed after 18-24 h long enrichments. In this study, we evaluated short enrichments (0, 2, 4, and 6 h) combined with a colorimetric loop-mediated isothermal AMPlification (LAMP) or real-time PCR to detect Salmonella and Campylobacter in poultry meat contaminated at different concentration levels (101, 103, and 105 CFU/g). Our results show that real-time PCR allows the detection of Salmonella and Campylobacter, even after shorter enrichment times than prescribed by ISO references; particularly, it detected Salmonella down to 101 CFU/g since T0 and Campylobacter from 103 CFU/g since T0. Detection with LAMP was comparable to real-time PCR without the requirement of a thermal cycler and with shorter execution times. These characteristics make colorimetric LAMP a valid alternative when one-day results are needed, improving the timely identification of positive meat batches, even in the absence of specialized instrumentation.

Modeling the behavior of Listeria innocua in Italian salami during the production and high-pressure validation of processes for exportation to the U.S.

A model describing Listeria innocua evolution according to process parameters of 51 Italian salami processes and HPP in 31 companies was developed. A total of 51 challenge tests were performed. During processing a L. innocua reduction of 0.34-4.32 Log10 CFU/g was observed and HPP further reduced the count of 0.48-3.47 Log10 CFU/g; an overall reduction of 1.04-5.68 is reached. PH after acidification/drying process, aw after seasoning, duration of the seasoning and caliber resulted associated (p < 0.05) with L. innocua decrease. HPP efficacy was associated (p < 0.05) with aw and pH of the product: higher the pH and aw after the acidification/drying and seasoning phases, higher resulted the L. innocua reduction after HPP. No significant association was observed between L.innocua and salt, nitrate and starter content and other characteristics of process. The model meets companies and Authorities needs and represents a useful tool to predict L. monocytogenes lethality, giving recommendations to food business operators interested in exportation to the U.S.

Raw donkey milk versus raw cow's milk. A preliminary study to compare the growth of Listeria monocytogenes and Staphylococcus aureus.

Gram-positive foodborne pathogens such as Listeria monocytogenes and Staphylococcus aureus can grow in a wide variety of foods, including raw milk. The aim of the study was to compare the growth of L. monocytogenes and S. aureus inoculated in donkey and cow samples of raw milk during a storage time of 11 days at 8 °C. Moreover, the study aimed to evaluate the influence of lactic acid bacteria (LAB) content on the growth of the two microbiological populations considered. LAB content was lower in raw donkey milk than in raw cow's milk during the entire analyses; on the other hand, pH levels were higher in the donkey milk rather than in the cow's milk, although both values showed a decrease at the day 11. S. aureus showed no significant differences in the two types of milk. From day 0 to 11, L. monocytogenes increased from 3.68 ± 0.02 log CFU/mL to 6.31 ± 0.07 log CFU/mL and from 3.64 ± 0.04 log CFU/mL to 4.59 ± 1.04 log CFU/mL, in donkey milk and in cow's milk, respectively. Our results showed that donkey milk is a more favourable matrix to support the growth of L. monocytogenes than cow's milk.

Behavior of Listeria innocua during the manufacturing and pit-ripening of Formaggio di Fossa di Sogliano PDO cheese.

Formaggio di Fossa di Sogliano is a traditional Italian Protected Designation of Origin (PDO) cheese ripened for a minimum of 5 months, with the feature of a ripening of at least 80 to at most 100 days in pits, digged into tuffaceous rocks according to medieval tradition of Italy. In this study, a challenge test using Listeria innocua as a surrogate of Listeria monocytogenes was performed, with the aim of increasing knowledge concerning the impact of the Fossa cheese process, and especially of the traditional ripening process of this PDO, on the behaviour of L. monocytogenes. Pasteurized milk was experimentally inoculated with 4.5 log CFU/mL cocktail by three L. innocua strains, and L. innocua and Mesophilic Lactic Acid Bacteria (LAB) counts as well as the evolution of temperatures, pH and aw values were monitored throughout the manufacturing and ripening processes. Throughout the ripening in maturation room a constant temperature of 8°C was observed reaching a temperature between 10 and 15.5°C during ripening into pit. In the final products data for LAB concentration, pH and aw values were roughly in accordance with literature, even if some differences were, probably due to variability of artisanal cheese productions. The numbers of L. innocua showed a slight decrease but remained stable until the end of ripening in maturation room, whereas a significant reduction of the microorganism was observed in the final product, at the end of the ripening into the pit. The findings give scientific evidence that the process of this PDO prevented the L. innocua growth, allowing us to speculate a similar behaviour of L. monocytogenes. Based on this study, the recommendation to extend as much as possible the ripening into pit (from 80 to 100 days) was provided to food business operators as a risk mitigation strategy to be implemented.

Reduction of Salmonella spp. populations in Italian salami during production process and high pressure processing treatment: Validation of processes to export to the U.S.

This study involved ten enterprises producing Italian salami, 20 different samples of fermented sausages underwent challenge tests to assess and record the following parameters: time, temperature, pH, aw, and Salmonella counts. A linear regression model was used to describe the Salmonella spp. decay: at the end of the process the result of total Salmonella reduction was 0.97-5.84 Log10 CFU/g and it was significantly associated with pH at the end of acidification/drying process, aw at the end of seasoning period, the duration of seasoning, and the caliber of salami respectively. High Pressure Processing (HPP) further reduced the Salmonella level by 2.41-5.84 Log10 CFU/g with an efficacy that resulted inversely associated with aw of salami at the end of seasoning; the objective of 5-Log reduction was always reached in all the cases tested by the production process plus HPP. This model could be a useful tool for enterprises and Authorities to evaluate the efficacy of the processes to reduce Salmonella load for exportation to the U.S.

Listeria monocytogenes in Gorgonzola cheese: Study of the behaviour throughout the process and growth prediction during shelf life.

As reported on RASFF's portal, in the first 9months of 2016, a total of 13 "alerts/information for attention" were issued concerning the presence of Listeria monocytogenes in mould cheeses throughout Europe. This study analyzes the behaviour of L. monocytogenes in Gorgonzola cheese, a typical Italian soft blue-veined cheese, when contaminated at different time points. In the first challenge test, the pasteurized milk was contaminated and the complete cheese manufacture (cheesemaking, ripening) and shelf life was simulated. After a decrease during the first days of the cheesemaking, the pH remained constant for 35days (5weeks) and then it increased rapidly reaching the final values of 6.8±0.02 in the core and 5.8±0.4 on the rind. At the same time, the pathogen concentration decreased (about 2logCFU/g), although during the last week a rapid pathogen growth was observed after the rise in pH values. When the cheese was stored at thermal abuse condition (8-12°C), the pathogen concentration on the rind was 4.8±0.3 log CFU/g and after 66days (about 9weeks) no significant difference (p>0.05) was observed; whereas, a growth from 5.4±0.4 to 7.1±0.5logCFU/g was observed in the core. A second challenge test was performed using three batches of commercial slices of Gorgonzola cheese inoculated by L. monocytogenes and stored at 8°C. The maximum specific growth rates (µmax, 1/h) of L. monocytogenes estimated ranged from 0.007 to 0.061. The square root model was used to predict the µmax at others temperature and to establish the time necessary to reach the European critical legal limit of 2logCFU/g, in different storage scenarios. The predictions obtained in this study can be applied to any time-temperature profile, and in particular to the conditions to which the product is most likely to be subject in normal use, up to its final consumption. This study can be considered a valuable contribution also aimed at supporting the monitoring surveys carried out by officers of the Regional Veterinary Authority.

Behaviour of Listeria Monocytogenes and Escherichia Coli O157:H7 During the Cheese Making of Traditional Raw-Milk Cheeses from Italian Alps.

The behaviour of Listeria monocytogenes and Escherichia coli O157:H7 was studied during the manufacture and ripening of two traditional Italian Alps cheeses. Each cheese type was manufactured in a pilot plan from raw cow milk (without the addition of starter cultures) artificially inoculated with L. monocytogenes and E. coli O157:H7 to a final concentration of about 4 log CFU/mL. The pathogens were enumerated throughout the cheese making and ripening processes to study their behaviour. When the milk was inoculated with 4 Log CFU/mL, the pathogens counts increased in the first time during the manufacturing process and then remained constant, until the end of ripening, or decreased significantly. Results indicate that the environment and nature of food borne pathogens affected the concentration of the bacteria during the manufacturing and ripening process. Thus, the presence of low cells numbers of L. monocytogenes and E. coli O157:H7 in milk destined for the production of raw milk cheeses characterized by a cooking of the curd less than 48°C can constitute a hazard for the consumer.

Microbiological and Physico-Chemical Changes During Manufacture of an Italian Goat Cheese Made from Raw Milk.

The aim of this work was to study the microbiological and physico-chemical changes throughout three cheesemaking replicates of Italian Formaggelle di capra cheese made from raw goat milk. Therefore, during the process, three samples of milk, curd and cheese at 3, 7, 11, 14, 21 and 30 days of ripening old cheese were taken from three cheesemaking replicates. The average of total mesophilic bacteria and Enterobacteriaceae count in raw milk was 5.27±0.57 and 3.8±1.02 Log cfu/mL, respectively. Lactic acid bacteria was the predominant bacterial group during the process, and they developed in different ways in each of the media used (M17 and MRS agar). Variability of microbial concentrations was observed between three cheesemaking replicates. A correlation between the presence of higher levels of Enterobacteriaceae in milk and the presence of other contaminants bacteria such as Escherichia coli ß-glucuronidase-positive and coagulase-positive staphylococci was observed. In cheesemaking replicate n. 2, E. coli level was 5.07±0.03 Log cfu/mL and increased by about 1 log until the last week of ripening, when the level decreased to 5.69±0.2 Log cfu/mL. The milk used for the cheesemaking replicate n. 2 was found to be contaminated also by coagulase-positive staphylococci (3.18±0.06 Log cfu/mL), but the behaviour of this group appeared to be very variable. In this study a first step of process control and microbial groups study was performed and the cheesemaking process was registered in the website www.ars-alimentaria.it, the Italian site supported by the Italian Board of Health.

Reduction of Escherichia Coli O157:H7 During Manufacture and Ripening of Italian Semi-Dry Salami.

In order to simulate a contamination at the processing plant, one batch of freshly-processed salami batter (20 kg) was inoculated (1% v:w) with 5 log colony forming unit (CFU)/g of a multi-strain cocktail of two strains of Escherichia coli O157:H7 (registered and wild strain). Another batch was inoculated (1% v:w) with sterile physiological saline solution and used to check the lactic acid bacteria (Lab) behaviour and the changes of physicochemical parameters (pH and aw ). Both batches were then processed to obtain a semi-dry salami (Hungarian-style): microbiological and physico-chemical properties were monitored during 94 days of ripening. During the manufacturing process, the levels of pathogen decreased of about 2.18 log CFU/g with respect to the initial inoculated levels. The behaviour of the indigenous bacteria such as Lab and the physico-chemical properties can help to determine the fate of pathogens throughout processing.

Study of Growth Potential of Listeria Monocytogenes in Low Fat Salami: An Innovative Italian Meat Product.

In the last years, consequently to EC Regulation no. 1924/2006 on nutrition and health claims made on foods, some Italian food businnes operators (FBOs) leaders in the meat sector, invested in research to develop innovative products such as low fat salami, containing up to 30% less fat than the traditional one. For FBOs it is essential to demonstrate for each production process whether the substrate allows the growth of L. monocytogenes and whether L. monocytogenes could reach or exceed the limit of 100 cfu g-1 at the end of the shelf life, as stated by EC Regulation no. 2073/2005. In the present study, the growth potential of L. monocytogenes during the shelf life of low fat salami packed in modified atmosphere was evaluated. The results show that the product is unable to support the growth of pathogen, even if the storage temperature is between 8 and 12°C.

Growth Potential of Listeria Monocytogenes in Sliced Turkey Bresaola Packed in Modified Atmosphere.

According to EC Regulation No 2073/2005, for food business operators that produce ready-to-eat (RTE) product, it is crucial to be able to demonstrate if the product supports the growth of Listeria monocytogenes. The objective of the study was therefore to evaluate the behaviour of L. monocytogenes in sliced RTE turkey bresaola (made by cured turkey breast 4.5% NaCl, 1% sodium lactate, sodium nitrite 150 ppm and flavouring) during the shelf life of the product, simulating a contamination during the slicing operation. Considering a shelf life of 90 days, as defined by manufacturer, the packages of sliced bresaola were stored at 5°C for 7 days and at 8°C for the remaining storage time (83 days). L. monocytogenes count decreased during storage test from 1.43/1.98 log cfu/g in the three batches tested to 1.03 log cfu/g in one batch and to undetectable levels in the other two batches. The results show that the investigated product is unable to support the growth of L. monocytogenes.

Behaviour of Escherichia Coli O157:H7 During the Manufacture and Ripening of an Italian Traditional Raw Goat Milk Cheese.

Formagelle di capra is a raw goat cheese produced from whole chilled goat milk; traditional technology involving unpasteurised milk and indigenous lactic starter cultures is employed for its production in Italy. The purpose of this study was to assess the behaviour of Escherichia coli O157:H7 during the manufacturing and ripening of this raw goat milk cheese. Raw milk was experimentally inoculated with E. coli O157:H7 in a laboratory scale plant and the count was monitored during production and 30 days of ripening required for this cheese. Results showed that E. coli O157:H7 count increased to more than 1.5 Log cfu g-1 during cheese production and remained constant until the end of ripening. The evidence that E. coli O157:H7 is able to survive during the manufacturing and ripening process suggests that the 30-day ripening period alone is insufficient to eliminate levels of viable E. coli O157:H7 in Formaggelle di capra cheese and that the presence of low numbers of E. coli O157:H7 in milk destined for the production of raw goat milk cheeses could represent a potential source of infection for humans and a threat for consumers.

Modelling Salmonella concentration throughout the pork supply chain by considering growth and survival in fluctuating conditions of temperature, pH and a(w).

We aim to predict the population density of Salmonella spp. through the pork supply chain under dynamic environmental conditions (pH, a(w) and temperature) that fluctuate from growth to survival/slow inactivation. To do this, the dependence of the probability of growth, and of the growth and inactivation rate on the temperature, pH and a(w) were modelled. Probabilistic and kinetic measurements, i.e. growth and survival curves, were collected from the ComBase database (www.combase.cc). Conditions at which selected data used to fit the models were generated covered wide ranges that are relevant to the pork supply chain. Probabilistic and kinetic models were combined to give predictions on the concentration of Salmonella spp. at any stage of the pork supply chain under fluctuating pH, a(w) and/or temperature. Models were implemented in a user-friendly computing tool freely available from http://www.ifr.ac.uk/safety/SalmonellaPredictions/. This program provides estimates on the population dynamics of Salmonella spp. at any stage of the pork supply chain and its predictive performance has been validated in several pork products.